of April 11, 2019.This information is current as Receptor 4

Inflammatory Responses through Toll-Like Filamentous Actin Reorganization, andInvolved in Bacterial Phagocytosis,

IsCoxiella burnetiiLipopolysaccharide from

Hubert Lepidi, Didier Raoult and Jean-Louis MegeCapo, Rudolf Toman, Shizuo Akira, Osamu Takeuchi, Amélie Honstettre, Eric Ghigo, Alix Moynault, Christian

http://www.jimmunol.org/content/172/6/3695doi: 10.4049/jimmunol.172.6.3695

2004; 172:3695-3703; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/6/3695.full#ref-list-1

, 26 of which you can access for free at: cites 43 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2004 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

Lipopolysaccharide from Coxiella burnetii Is Involved inBacterial Phagocytosis, Filamentous Actin Reorganization, andInflammatory Responses through Toll-Like Receptor 41

Amelie Honstettre,* Eric Ghigo,* Alix Moynault,* Christian Capo,* Rudolf Toman,†

Shizuo Akira,‡ Osamu Takeuchi,‡ Hubert Lepidi,* Didier Raoult,* and Jean-Louis Mege2*

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innateimmune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains un-known. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macro-phages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not inthat of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages fromTLR4�/� mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which wasprevented in TLR4�/� macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4�/� macrophages,suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages.These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii weresimilar in wild-type and TLR4�/� mice. We also showed that the number of granulomas was decreased in the liver of infectedTLR4�/� mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associatedwith decreased production of IFN-� and TNF. Taken together, these results demonstrate that TLR4 controls early events of C.burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production. The Journal of Immunology,2004, 172: 3695–3703.

T he recognition of microbial pathogens by the innate im-mune system is critical for the activation of microbicidaleffectors and the orientation of adaptive immunity (1).

Toll-like receptors (TLRs),3 a molecular family conservedthroughout the evolution, act as major sensors of infectious non-self and as receptors for the activation of innate immune system(2). Recent extensive literature has demonstrated the specificity ofsome TLRs including TLR4 and TLR2 toward microorganisms ortheir products. Indeed, TLR4 recognizes LPS and Gram-negativebacteria such as Escherichia coli and Salmonella sp., whereasTLR2 is mainly involved in the recognition of lipoproteins, pro-teoglycans, lipopeptides, Gram-positive bacteria, and mycobacte-ria (2, 3). Most of the evidence concerning the role of TLRs ininnate response to bacteria has been obtained with extracellularbacteria and facultative intracellular bacteria. Their role in the rec-ognition of obligate intracellular bacteria remains unknown.

Coxiella burnetii, the etiologic agent of Q fever, is an obligateGram-negative bacterium that inhabits monocytes/macrophages(4). Only LPS has been associated with bacterial virulence. Indeed,antigenic variation similar to smooth-to-rough variation of enter-obacteria has been described in C. burnetii: bacteria in phase I(virulent organisms) express a smooth-type LPS (phase I LPS)whereas bacteria in phase II (avirulent organisms) exhibit a rough-type LPS (phase II LPS) (5). Phase II LPS is truncated and lacksthe branched-chain sugars virenose and dihydrohydroxystreptosepresent in phase I LPS (6). The antigenic phase transition of LPSis associated with a chromosomal deletion (7); this concerns alarge group of LPS biosynthetic genes arranged in O-Ag clusterincluding genes involved in virenose synthesis (8). The role of LPSin the pathogenicity of C. burnetii is poorly understood. It plays arole in bacterial immunogenicity and induces a strong Ab response(9). Although C. burnetii LPS is considered as poorly endotoxic incontrast to LPS from enterobacteria (10), it induces the production ofinflammatory cytokines in murine and human macrophages (11, 12).We have previously found that TNF production by human macro-phages is differentially induced by phase I and phase II LPSs (12).

C. burnetii virulence depends on the ability of organisms toenter macrophages and to escape from their microbicidal activity.Indeed, virulent organisms are poorly internalized and survive inhuman monocytes, whereas avirulent variants are efficiently inter-nalized but are eliminated. The uptake of virulent C. burnetii de-pends on �v�3 integrin whereas that of avirulent bacteria requires�v�3 integrin and CR3 (�M�2 integrin) (13). The control of C.burnetii phagocytosis results from the inappropriate activation ofhost cells. Virulent organisms, but not avirulent organisms, stim-ulate the activation of Lyn and Hck, two src-related proteintyrosine kinases. Their activation leads to actin cytoskeleton reor-ganization involved in the impairment of phagocytosis of virulent

*Unite des Rickettsies, Centre National de la Recherche Scientifique Unite Mixte deRecherche 6020, Institut Federatif de Recherche 48 Universite de la Mediterranee,Marseille, France; †Department of Rickettsiology and Chlamydiology, Institute ofVirology, Slovak Academy of Sciences, Bratislava, Slovak Republik; and ‡Depart-ment of Host Defense, Research Institute for Microbial Diseases, Osaka University,Osaka, Japan

Received for publication May 16, 2003. Accepted for publication January 8, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Programme Hospitalier de Recherche Clinique2001.2 Address correspondence and reprint requests to Dr. Jean-Louis Mege, Unite desRickettsies, Faculte de Medecine, 27 boulevard Jean Moulin, 13385 Marseille Cedex5, France. E-mail address: Jean-Louis.Mege@medecine.univ-mrs.fr3 Abbreviations used in this paper: TLR, Toll-like receptor; LPC, lysophosphatidyl-choline; F-actin, filamentous actin; Lamp, lysosome-associated membrane protein;PG, peptidoglycan; wt, wild type.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

C. burnetii (14, 15). We have recently demonstrated that the sur-vival of virulent C. burnetii inside monocytes is based on the im-paired fusion of bacterial phagosomes with lysosomes whereasphagosomes containing avirulent organisms undergo completephagosome-lysosome fusion (16).

In this study, we investigated the role of bacterial LPS andTLR4 in C. burnetii infection in vitro and in vivo. We showed thatphase I LPS, but not phase II LPS, was involved in the phagocy-tosis of organisms by macrophages through TLR4. In addition,phase I LPS induced filamentous (F)-actin reorganization, whichwas suppressed in TLR4�/� macrophages. In contrast, the inter-action of C. burnetii with TLR4 did not influence the intracellularsurvival of bacteria in macrophages and bacterial clearance invivo. TLR4 was involved in the formation of granulomas and theproduction of inflammatory cytokines in C. burnetii-infected mice.Although TLR4 is dispensable for late control of C. burnetii in-fection, it controls the early events of C. burnetii infection includ-ing macrophage phagocytosis, granuloma formation, and cytokineproduction.

Materials and MethodsMice, cells, and bacteria

TLR4�/� mice were generated on a mixed 129/Ola � C57BL/6 back-ground and backcrossed to C57BL/6 for seven generations as describedelsewhere (17). C57BL/6 mice (Charles River Breeding Laboratories,L’Arbresle, France) were used as control mice expressing TLR4 (wild-type, wt). C3H/HeN mice (that express functional TLR4) and C3H/HeJmice (that do not express functional TLR4) were purchased from CharlesRiver Breeding Laboratories. TLR2�/� mice were generated on C57BL/6background as described elsewhere (17). The human myelomonocytic cellline THP-1 was cultured in RPMI 1640 containing 25 mM HEPES, 10%FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 �g/ml streptomycin(Invitrogen, Eragny, France) by biweekly passages (15). Murine macro-phages were recovered by washing peritoneal cavity with ice-cold HBSS.They were obtained by adherence after a 2-h incubation and washing withHBSS to remove nonadherent cells. Virulent C. burnetii organisms (NineMile strain) were recovered from mouse spleens at 10 days postinfection,and were cultured in mouse L929 fibroblasts for two passages in antibiotic-free MEM (Invitrogen) supplemented with 4% FBS and 2 mM L-glutamine(13). Avirulent variants of C. burnetii were cultured in L929 cells by re-peated passages. After cell homogenization, virulent and avirulent bacteriawere purified on 25% to 45% linear Renografin gradients. The concentra-tion of C. burnetii was determined by Gimenez staining. Bacterial viabilitywas determined using the LIVE/DEAD BacLight bacterial viability kit(Molecular Probes, Eugene, OR), as previously described (18). Phase I andphase II LPSs were isolated from Nine Mile organisms, as described else-where (19, 20).

Bacterial phagocytosis and intracellular fate of C. burnetii

For phagocytosis study, THP-1 monocytes (5 � 105 cells per assay), pre-treated or not with 5 �g/ml polymyxin B (Sigma-Aldrich, St. Louis, MO)for 30 min, were incubated with C. burnetii (200:1 bacterium to cell ratio)in 0.5 ml RPMI 1640 at 37°C. After washing to remove free bacteria, cellswere centrifuged at 800 � g. Adherent murine macrophages (2 � 105

cells/assay) were incubated with C. burnetii (200:1 bacterium to cell ratio)in 1 ml RPMI 1640 at 37°C. In some experiments, they were pretreatedwith 50 ng/ml peptidoglycan (PG) from Staphylococcus aureus (Sigma-Aldrich) for 30 min, incubated with organisms in the presence of PG for4 h, and washed to remove unbound bacteria. Then, THP-1 monocytes andmurine macrophages were fixed with 4% paraformaldehyde, and bacteriawere revealed by immunofluorescence as previously described (13).Briefly, cell preparations were incubated with rabbit Abs directed againstC. burnetii (at 1/250 dilution) in the presence and the absence of 0.1 mg/mllysophosphatidylcholine (LPC; Sigma-Aldrich), washed, and incubatedwith 1/200 dilution of FITC-conjugated F(ab�)2 anti-rabbit IgG (BeckmanCoulter, Roissy, France). Without LPC, only cell-bound organisms wererevealed; bound and ingested organisms were revealed in the presence ofLPC. The association index was quantified as follows: [(number of bacteriaper positive cell) � (percentage of positive cells)] � 100. The difference ofindexes in the presence or absence of LPC quantified the uptake of C.burnetii (phagocytosis index).

For the follow-up of the infection, adherent macrophages were incu-bated with C. burnetii (200:1 bacterium to cell ratio) for 4 h at 37°C. Theywere then washed to remove free bacteria; this time point was designatedas day 0. Infected macrophages were cultured for 6 days, and bacteria wererevealed by indirect immunofluorescence as previously described. Resultsare expressed as infection index that assesses the number of bacteria perpositive cell and the percentage of positive cells � 100.

F-actin analysis by laser scanning confocal fluorescencemicroscopy

The intracellular distribution of F-actin was determined as follows: THP-1cells (5 � 105 cells/assay) or adherent macrophages (2 � 105 cells/assay)were stimulated with C. burnetii (200:1 bacterium to cell ratio) or C. bur-netii LPS in the presence and the absence of polymyxin B, and fixed with4% paraformaldehyde. After permeabilization with 0.1 mg/ml LPC inHBSS, cells were incubated with 10 U/ml bodipy phallacidin (MolecularProbes) for 20 min. The specimens were examined with a laser scanningconfocal fluorescence microscope (TCS 4D; Leica, Heidelberg, Germany)equipped with a 100� (NA 1.4) oil immersion lens, as previously de-scribed (14). Serial optical sections of images were collected at 0.5 �mintervals and analyzed with Adobe Photoshop V.5.5 (Adobe Systems,Mountain View, CA).

Intracellular traffic of C. burnetii

Adherent murine macrophages (2 � 105 cells/assay) were incubated withC. burnetii (200:1 bacterium to cell ratio) for different periods of time.After being washed, cells were fixed with 4% paraformaldehyde, and freealdehydes were quenched with 0.5 M ammonium chloride, as previouslydescribed (21). Cells were permeabilized by 0.1% saponin (Sigma-Aldrich)in PBS containing 10% horse serum (Invitrogen) for 30 min and washed.They were then incubated for 30 min with PBS containing 0.1% saponin,5% horse serum, 1/2000 dilution of human Abs to C. burnetii, and rabbitAbs specific for lysosome-associated membrane protein (Lamp)-1 or ca-thepsin D (1/50 and 1/500 dilutions, respectively; BD Transduction Lab-oratories, Lexington, KY). After washing, cells were incubated with 1/100dilution of Texas Red-conjugated anti-human IgG Abs and FITC-conju-gated anti-rabbit IgG Abs (Beckman Coulter) for 20 min. Thereafter, cellswere washed, mounted with Mowiol (Calbiochem, San Diego, CA), andstored at 4°C until examination. The colocalization of organisms with in-tracellular markers was examined with a laser scanning confocal fluores-cence microscope equipped with suitable filters, as described elsewhere(16). Briefly, optical sections of images (1 �m) were analyzed using AdobePhotoshop V5.5. Vacuoles containing C. burnetii were scored as positivefor soluble cathepsin D when fluorescence was observed in the phagosomelumen; for Lamp-1, a membrane marker of late endosomes, vacuoles werescored as positive when a fluorescence ring surrounded organisms. Ap-proximately 30 vacuoles containing C. burnetii were scored per coverslip,and at least three distinct experiments were performed per condition. Re-sults are expressed as the percentage of phagosomes that colocalized withintracellular markers.

Determination of in vivo infection

TLR4�/� and wt mice, aged 5–6 wk, were challenged i.p. with 5 � 105

virulent C. burnetii organisms. They were sacrificed 7, 14, and 21 daysafter infection, and liver, spleen, heart, lungs, and mesenteric lymph nodeswere excised, and blood samples were collected for serology. The organs,fixed in 10% neutral-buffered formalin, were sectioned and embedded inparaffin. For each tissue specimen, serial 5-�m thick sections were ob-tained to perform H&E staining and immunohistochemical investigations.The granuloma expression was assessed by light microscopy. Granulomaswere defined as collections of 10 or more macrophages and lymphocyteswithin the organs. The number of granulomas was determined after wholeexamination of at least three tissue sections of each organ. The detection ofC. burnetii organisms in tissues was performed by immunohistochemistryon serial deparaffinized sections of all organs as previously described (22).Briefly, each tissue section was incubated with anti-C. burnetii rabbit Abs(at 1/2000 dilution) or normal rabbit serum as control. Immunodetectionwas performed with biotinylated anti-rabbit Abs and peroxidase-labeledstreptavidin (Zymed CliniSciences, Montrouge, France) with amino-ethyl-carbazole as substrate. After washing, slides were counterstained withMayer’s hematoxylin for 5 min and the bacteria were visualized in tissuesas precipitation products. The whole number of bacteria detected within thegranulomas by light microscopy was determined for each tissue section.

The surface area of tissue sections microscopically examined was de-termined by quantitative image analysis, as previously described (23). Im-ages of tissue sections were acquired using a camera (Sony, Paris, France)

3696 ROLE OF TLR4 IN C. burnetii PHAGOCYTOSIS AND HOST RESPONSE

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

mounted on a Zeiss Axiophot/DMX1200 microscope (Zeiss, Rueil Mal-maison, France). Histological images were digitized with an automatedimage analysis system (SAMBA Technologies, Alcatel TITN, Grenoble,France). For each study, a specific interactive program providing control ofanalysis was developed to measure tissue section surface on each slide. Thenumber of granulomas and organisms within each granuloma was ex-pressed per tissue in square millimeters.

The presence of Abs to C. burnetii in serum from wt and TLR4�/� micewas determined by microimmunofluorescence, as described elsewhere(24).

Cytokine determination

Splenocytes and adherent macrophages from wt and TLR4�/� mice wereincubated in RPMI 1640 containing 25 mM HEPES, 10% FBS, 2 mML-glutamine, 100 U/ml penicillin, and 100 �g/ml streptomycin (Invitro-gen). All media were checked for the absence of endotoxins with Limulusamebocyte lysate (Cambrex Bioscience, Emerainville, France). Spleno-cytes (2 � 106 cells in 1 ml) were incubated in flat-bottom 24-well cultureplates (Nunc, PolyLabo, Strasbourg, France) with or without heat-inacti-vated C. burnetii (10 organisms per cell) for 24 h at 37°C. Once collected,supernatants were stored at �80°C until IFN-� determination. Adherentmacrophages (106 cells/assay) were stimulated by heat-inactivated C. bur-netii (10 organisms per cell) for 24 h at 37°C, and cell supernatants weretested for the presence of TNF and IL-10. The three cytokines were mea-sured by immunoassays. IFN-� (detection limit: 10 pg/ml) and IL-10 (de-tection limit: 12 pg/ml) assays were provided by Endogen (BioAdvance,Emerainville, France). TNF (detection limit: 5 pg/ml) assay was from R&DSystems (Abingdon, U.K.). The intra- and interspecific coefficients of vari-ation were �10%.

Statistical analysis

Results, given as the mean � SD, were compared with Student’s t test.Differences were considered significant at p � 0.05.

ResultsLPS from virulent C. burnetii is involved in bacterial uptakethrough TLR4

As virulent and avirulent C. burnetii organisms are differently in-ternalized by monocytes and exhibit distinct LPS structures, weinvestigated the role of LPS in phagocytosis by incubating THP-1monocytes with polymyxin B, known to interfere with LPS bind-ing. In the absence of serum, polymyxin B (at 5 �g/ml) decreasedthe uptake of virulent C. burnetii by 35% after 2 h ( p � 0.03) andby 50% after 4 h ( p � 0.02), but it had no effect on the uptake ofavirulent organisms (Table I). In the presence of AB serum as asource of LPS-binding protein, the uptake of virulent and avirulentC. burnetii was higher than in its absence. Again, polymyxin Binhibited the internalization of virulent organisms by 55% after 2 h( p � 0.01) and by 60% after 4 h ( p � 0.02), without affecting theuptake of avirulent organisms. The role of LPS in C. burnetii up-take was not restricted to THP-1 monocytes because polymyxin Binhibited the uptake of virulent C. burnetii by monocytes isolatedfrom peripheral blood to a similar extent (data not shown). AsTLR4 is involved in the recognition of several species of LPS (25),

we investigated the role of TLR4 in the uptake of C. burnetii usingperitoneal macrophages from wt and TLR4�/� mice (Fig. 1). Thephagocytosis of virulent organisms by macrophages from wt micewas lower than that of avirulent organisms, as described for THP-1cells. The uptake of virulent C. burnetii was decreased by �40%in macrophages from TLR4�/� mice with or without addition ofmouse serum ( p � 0.05). The decrease in uptake of virulent C.burnetii was similar in macrophages from TLR4�/� mice and inTHP-1 monocytes in the presence of polymyxin B (compare Fig.1 and Table I). In contrast, the uptake of avirulent organisms wassimilar in wt and TLR4�/� macrophages (Fig. 1). C. burnetiiphagocytosis was also studied in C3H/HeJ mice that do not ex-press functional TLR4. The uptake of virulent C. burnetii by peri-toneal macrophages was decreased by 45% in C3H/HeJ mice com-pared with C3H/HeN mice whereas the uptake of avirulentorganisms was not affected by the lack of functional TLR4. Hence,the phagocytosis of virulent C. burnetii by macrophages involvesbacterial LPS and TLR4, in contrast to that of avirulent organisms.

FIGURE 1. C. burnetii phagocytosis by TLR4�/� macrophages. Mac-rophages (2 � 105 cells/assay) from wt and TLR4�/� mice were incubatedwith virulent and avirulent C. burnetii (200:1 bacterium to cell ratio) in thepresence or the absence of 10% mouse serum for 4 h at 37°C. Bacteria weredetected by immunofluorescence. Results are expressed as phagocytosisindex and are the mean � SE of four experiments. �, p � 0.05 representsthe comparison between wt and TLR4�/� macrophages.

Table I. Effect of polymyxin B on C. burnetii phagocytosisa

Polymyxin B (h)

Without AB Serum With AB Serum

� � � �

Virulent C. burnetii (2) 60 � 7 39 � 3* 153 � 17 69 � 5***Virulent C. burnetii (4) 124 � 10 62 � 4** 337 � 70 134 � 35**Avirulent C. burnetii (2) 179 � 27 216 � 42 329 � 48 389 � 36Avirulent C. burnetii (4) 295 � 39 354 � 35 653 � 35 590 � 46

a THP-1 monocytes (5 � 105 cells/assay) were pretreated with polymyxin B (5 �g/ml) and then incubated with C. burnetii(200:1 bacterium to cell ratio) in the presence or absence of 10% human AB serum for 2 and 4 h. Bacteria were detected byimmunofluorescence. Results are expressed as phagocytosis index and are the mean � SE of five experiments.

* p � 0.03; **p � 0.02; ***p � 0.01, which represent the comparison of C. burnetii uptake by THP-1 cells in the presenceand the absence of polymyxin B.

3697The Journal of Immunology

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

LPS from virulent C. burnetii stimulates F-actin reorganizationthrough TLR4

As the phagocytosis of virulent C. burnetii is associated with theformation of pseudopodal extensions and polarized distribution ofF-actin (14), we investigated the involvement of bacterial LPS andTLR4 in cytoskeleton reorganization. In macrophages from wtmice, virulent C. burnetii induced cell spreading and the formationof polarized filopodia and lamellipodia. F-actin was concentratedbeneath filopodia and lamellipodia and as spots in cytoplasmicareas. In contrast, avirulent variants of C. burnetii had no effect onF-actin organization (Fig. 2A). Phase I LPS reproduced C. bur-netii-induced morphological changes of macrophages consisting ofcell spreading, filopodia, polarized lamellipodia and cytoplasmicspots of F-actin (Fig. 2A). After 10 min of stimulation with 1�g/ml phase I LPS, �80% of cells exhibited filopodia, and thepercentage of cells with filopodia decreased thereafter (Fig. 2B).Filopodia were detected in response to 0.25 �g/ml phase I LPS andthe number of cells with filopodia became maximum with 1 �g/mlphase I LPS (Fig. 2C). In contrast to phase I LPS, phase II LPS didnot stimulate the formation of filopodia and F-actin reorganization(Fig. 2A) whatever the time of stimulation and the dose of LPS. Inmacrophages from TLR4�/� mice, the reorganization of F-actinand the formation of filopodia stimulated by virulent C. burnetiiand phase I LPS were prevented (Fig. 2). Similarly, the cytoskel-eton reorganization induced by virulent C. burnetii was decreasedby 75% in macrophages from C3H/HeJ mice compared with mac-rophages from C3H/HeN mice. Hence, C. burnetii-stimulated mor-phological changes and F-actin reorganization require bacterialLPS and TLR4.

TLR4 is not involved in intracellular survival of C. burnetii

As the interaction of virulent C. burnetii with TLR4 is critical forearly events associated with bacterial uptake, we investigated itsrole in C. burnetii survival. Macrophages from wt and TLR4�/�

mice were incubated with virulent and avirulent organisms for 4 h,and infected cells were cultured for 6 days. In wt macrophages

infected with virulent C. burnetii, the infection index slowly de-creased at day 3 postinfection and remained constant thereafter(Fig. 3A). Intracellular bacteria remained viable during the courseof the experiments as assessed by measuring C. burnetii viability(data not shown). In contrast, the number of avirulent organismsmarkedly decreased after 3 days (80% inhibition) and the infectionindex was minimum at day 6 postinfection. The decreased numberof bacteria corresponded to their killing, as assessed by decreasedviability (data not shown). In TLR4�/� macrophages infected withvirulent C. burnetii, the infection index was similar to that foundin wt macrophages at days 3 and 6 postinfection, and the bacterialviability was not modified. The infection index rapidly decreasedin TLR4�/� macrophages infected with avirulent variants of C.burnetii, as found in wt macrophages (Fig. 3A). Thus, the survivalof virulent C. burnetii organisms in macrophages does not dependon their interaction with TLR4.

As the survival of virulent C. burnetii is associated with im-paired fusion of phagosomes with lysosomes in monocytes (16), C.burnetii trafficking was investigated in TLR4�/� macrophages.For that purpose, the colocalization of C. burnetii phagosomeswith Lamp-1, a marker of late endosomes-lysosomes, and cathep-sin D, a lysosomal protease, was determined. In wt and TLR4�/�

macrophages incubated with virulent or avirulent C. burnetii (day0), �70% of phagosomes colocalized with Lamp-1, which ap-peared as a ring surrounding the organisms. At day 2 postinfection,all phagosomes had acquired Lamp-1 (Fig. 3B). In wt andTLR4�/� macrophages, cathepsin D appeared in the lumen of40% of phagosomes containing avirulent organisms at day 0. Thepercentage of phagosomes that colocalized with cathepsin Dsteadily increased thereafter and reached 60% at day 2 postinfec-tion. In contrast, phagosomes containing virulent C. burnetii wereunable to colocalize with cathepsin D whatever the time of postin-fection in wt and TLR4�/�macrophages (Fig. 3C). Hence, TLR4does not interfere with the trafficking of C. burnetii in murinemacrophages.

FIGURE 2. F-actin distribution in TLR4�/�

macrophages. A, Macrophages (2 � 105 cells/assay)from wt and TLR4�/� mice were stimulated by C.burnetii (200:1 bacterium to cell ratio) or C. burnetiiLPS (1 �g/ml) for 10 min at 37°C. F-actin was la-beled with 10 U/ml bodipy phallacidin. Cells wereexamined by laser scanning confocal microscopy,and representative cells are shown. B, Macrophagesfrom wt and TLR4�/� mice were stimulated withphase I LPS (1 �g/ml) for different periods of time.C, Macrophages from wt and TLR4�/� mice werestimulated with different concentrations of phase ILPS for 10 min. F-actin was labeled with bodipyphallacidin. The results are expressed as the percent-age of macrophages showing filopodia and representthe mean � SE of three experiments.

3698 ROLE OF TLR4 IN C. burnetii PHAGOCYTOSIS AND HOST RESPONSE

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

Role of TLR4 in C. burnetii infection in vivo

We wondered whether the lack of TLR4 may affect the suscepti-bility and/or the resistance of mice toward C. burnetii. TLR4�/�

and wt mice were i.p. injected with 5 � 105 virulent organisms,and their infection was recorded up to 21 days. Mortality or mor-bidity was not observed in wt and TLR4�/� mice, suggesting thatmice remained resistant to C. burnetii despite the lack of TLR4.The course of infection was assessed by measuring circulatingAbs. In wt mice, Abs directed against C. burnetii were detectedafter 7 days of infection and their titer reached a plateau after 14and 21 days. In TLR4�/� mice, the kinetics of production of spe-cific Abs was delayed but, after 14 days, Ab titers were similar inwt and TLR4�/� mice (Table II). The course of tissular infectionwas also assessed by immunodetection of C. burnetii. In wt andTLR4�/� mice, C. burnetii organisms were detected in liver and

spleen, whereas lung, heart, and mesenteric lymph nodes remaineddevoid of organisms. The organisms were found in intracellularlocation as coarse or fine grains, and immunopositive cells corre-sponded to macrophages inside granulomas (Fig. 4A). In wt andTLR4�/� mice, the organisms were detectable after 4 days in thespleen; bacterial load reached a peak at day 7 and it decreasedthereafter to become undetectable at day 21. In the liver from wtand TLR4�/� mice, C. burnetii organisms were detected at 4 dayspostinfection and their number steadily decreased to reach mini-mum amount at day 21 (Fig. 4B). Hence, bacterial clearance wasindependent of TLR4.

As C. burnetii organisms were detected within inflammatorygranulomas, we investigated the expression of granulomas in eachtarget organ. In wt mice, lesions appeared as aggregates mainly com-posed of macrophages and lymphocytes and few polymorphonuclear

FIGURE 3. C. burnetii survival and intracellular trafficking in TLR4�/� macrophages. Macrophages (2 � 105 cells/assay) were infected by C. burnetii(200:1 bacterium to cell ratio) for 4 h at 37°C, washed to remove free bacteria (considered as day 0), and incubated for 6 days. A, The infection index wasdetermined by indirect immunofluorescence. The results are expressed as relative to day 0. B and C, Organisms (top panels) and Lamp-1 or cathepsin D(middle panels) were revealed by indirect immunofluorescence and confocal microscopy. Paired photomicrographs show C. burnetii and Lamp-1 (B) orcathepsin D (C) after a 4-h incubation. Phagosomes (�100) were numerated in each experimental condition. Results (bottom panels) are expressed inpercentage � SE of phagosomes that express Lamp-1 (B) or cathepsin D (C).

3699The Journal of Immunology

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

leukocytes without suppuration. The granulomatous reactions werefocal and scattered throughout liver lobules, portobiliary spaces,and splenic red pulp (Fig. 5A). In the spleen of wt mice, granulo-mas were detected after 4 days; their number became maximizedbetween days 7 and 14 and decreased thereafter (Fig. 5B). In thespleen from TLR4�/� mice, the number of granulomas was sim-ilar to that of wt mice after 4 and 7 days, but it was significantlylower ( p � 0.02) than in wt mice at days 14 and 21 postinfection.In the liver from wt mice, the number of granuloma was high atday 4 and day 7 postinfection, and steadily decreased thereafter(Fig. 5B). In TLR4�/� mice, the number of liver granulomas wassignificantly lower ( p � 0.02) than in wt mice at days 4 and 7postinfection. The area of granulomas and the number of cellswithin the granulomas were significantly reduced in TLR4�/�

mice as compared with wt mice (Fig. 5C). Taken together, theseresults show that TLR4 is critical for the formation of granulomasin C. burnetii-infected mice.

Role of TLR4 in C. burnetii-stimulated production of cytokines

The role of TLR4 in the control of granuloma formation may resultfrom the modulation of the production of cytokines such as IFN-�and TNF, known to be required for granuloma formation. First,splenocytes from wt and TLR4�/� mice infected by C. burnetiiwere incubated with or without C. burnetii, and the release ofIFN-� was assessed. In the absence of stimulation, splenocytesfrom uninfected mice did not release IFN-�. IFN-� was producedby unstimulated wt splenocytes (1165 � 325 pg/ml) but not byunstimulated TLR4�/� splenocytes at day 7 postinfection. C. bur-netii-stimulated splenocytes from uninfected wt mice producedIFN-� to a low extent; the release of IFN-� dramatically increasedin splenocytes of wt mice infected for 7 days and remained highuntil day 21 (Fig. 6A). In stimulated TLR4�/� splenocytes, therelease of IFN-� was decreased by � 50% whatever the time ofinfection of mice, and it was almost undetectable at day 21 postin-fection. Second, the production of TNF was assayed in superna-tants from unstimulated macrophages. TNF was released by mac-rophages from uninfected wt mice (21 � 5 pg/ml) and its amountwas maximum at day 7 postinfection (71 � 18 pg/ml); it wasundetectable in TLR4�/� mice. C. burnetii-stimulated TNF pro-duction by macrophages from uninfected wt mice, and TNF pro-duction was dramatically increased in macrophages from infectedmice (Fig. 6B). C. burnetii-stimulated TNF production byTLR4�/� macrophages was decreased by 50% as well in unin-fected mice than as in infected mice. The partial effect of TLR4lack on TNF production was specific for C. burnetii because theTNF production stimulated by LPS from E. coli was depressed by�90% whatever the time of infection of TLR4�/� mice comparedwith wt mice (data not shown). Third, we investigated the produc-tion of IL-10, known to down-modulate inflammatory cytokines,by peritoneal macrophages. Unstimulated macrophages from wt

mice released IL-10 only at days 7 (59 � 18 pg/ml) and 14 (38 �6 pg/ml) postinfection. The unstimulated release of IL-10 was un-detectable in macrophages from TLR4�/� mice. In response to C.burnetii stimulation, IL-10 was produced by macrophages fromuninfected and infected wt mice. In macrophages from TLR4�/�

mice, C. burnetii-stimulated production was decreased by 80% atday 7 postinfection and by 50% at days 14 and 21 (Fig. 6C). Takentogether, these results show that TLR4 is involved in cytokinerelease in C. burnetii-infected mice.

Role of TLR2 in macrophage responses to C. burnetii

Because TLR4 was involved in C. burnetii phagocytosis, we askedwhether TLR2 was directly or indirectly involved in this response.First, we investigated the role of TLR2 in the phagocytosis of C.burnetii and the F-actin reorganization induced by virulent organ-isms. The uptake of virulent and avirulent C. burnetii was similarin wt and TLR2�/� mice (Fig. 7A). The lack of TLR2 did notaffect the F-actin reorganization stimulated by C. burnetii (Fig.7B). Second, we wondered whether TLR2 engagement may restorethe impaired phagocytosis of C. burnetii found in TLR4�/� mac-rophages. For that purpose, macrophages were pretreated with PG,a specific ligand of TLR2, for 30 min and then incubated withvirulent C. burnetii. The addition of PG to wt macrophages had noeffect on C. burnetii phagocytosis (Fig. 7C). In contrast, the addi-tion of PG to TLR4�/� macrophages significantly ( p � 0.05) in-creased C. burnetii phagocytosis to the level found in wt macro-phages. Taken together, these results suggest that TLR2 is notinvolved in C. burnetii phagocytosis but that its engagement res-cued impaired phagocytosis of TLR4�/� mice.

DiscussionThe aim of this report was to study the role of LPS in C. burnetiiinfection, an obligate intracellular organism. The composition ofLPSs from C. burnetii is distinct from that of LPSs isolated fromother Gram-negative microorganisms, which are highly endotoxic

Table II. Anti-C. burnetii Abs in wt and TLR4�/� micea

Postinfection Time (day)

Anti-C. burnetii IgG (Titer)

WT mice TLR4�/� mice

7 50 (50–100) 0 (0–0)14 800 (400–1600) 800 (400–800)21 1600 (800–1600) 800 (800–1600)

a WT and TLR4�/� mice were infected with 5 � 105 C. burnetii organisms andserum was collected after 7, 14, and 21 days. The presence of IgG directed against C.burnetii was assessed by microimmunofluorescence using C. burnetii in phase I. Theresults as titers are expressed as median with range (in parentheses) of five mice ateach time.

FIGURE 4. Immunodetection of C. burnetii. TLR4�/� and wt micewere infected by C. burnetii (5 � 105 organisms), and sacrificed at differenttimes. Bacteria were revealed by immunohistochemistry. A, Representativemicrographs of spleen and liver are shown. B, C. burnetii organisms werenumerated by image analysis. The results are expressed as the number ofbacteria per square millimeter of spleen (left) and liver (right), and are themean � SE of five mice per time point.

3700 ROLE OF TLR4 IN C. burnetii PHAGOCYTOSIS AND HOST RESPONSE

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

(6). LPS from virulent C. burnetii was involved in bacterial inter-nalization by monocytes/macrophages. Indeed, polymyxin B, aLPS antagonist, decreased the uptake of virulent C. burnetii bymonocytes. Nevertheless, the inhibition was only partial, suggest-ing that the role of phase I LPS in phagocytosis is complementaryto previously reported entry mechanisms. Indeed, virulent C. bur-netii is internalized by monocytes through the engagement of �v�3

integrin whereas the uptake of avirulent organisms engages both�v�3 integrin and CR3 through the activation of integrin-associ-ated protein (13). As the integrins including �v�3 integrin are un-able to support efficient binding and internalization in resting cellswithout activating signals, it is likely that phase I LPS mediates acostimulation signal enabling �v�3 integrin to acquire an activeconformation, and thus allows C. burnetii recognition and inter-nalization. This is reminiscent of the role of LPS in �2 integrinactivation (26). LPS-mediated activation of integrins may dependon their interaction with cytoskeleton (27, 28). We have previouslyreported that virulent but not avirulent C. burnetii organisms in-duce reorganization of F-actin in monocytes (15). C. burnetii-stim-ulated reorganization of F-actin was due to bacterial LPS. The LPSisolated from virulent C. burnetii induced the formation of filop-

odia and lamellipodia in murine macrophages, as did intact C.burnetii organisms. This property is not specific of C. burnetii LPSbecause LPS from enterobacteria, known to be highly endotoxic,induces similar reorganization of cytoskeleton in monocytes andmacrophages (29). The role of LPS in C. burnetii phagocytosisdepends on its effect on actin cytoskeleton, which controls bacte-rial receptor redistribution and orientates C. burnetii toward theleading edge of host cells (30).

The effect of LPS from virulent C. burnetii on bacterial phago-cytosis and F-actin reorganization is mediated by TLR4. To ourknowledge, this is the first demonstration of TLR4 involvement inearly cellular events associated with the phagocytosis process. Theuptake of virulent organisms was decreased in TLR4�/� macro-phages as compared with wt macrophages, as observed in the pres-ence of polymyxin B. This result did not depend on the geneticbackground of mice because similar results were obtained inTLR4�/� and C3H/HeJ macrophages. It demonstrates that LPSdistinct from canonical LPS isolated from enterobacteria may in-teract with TLR4. In contrast, the uptake of avirulent variants of C.burnetii was not impaired in TLR4�/� macrophages, suggestingthat the interaction of avirulent bacteria with macrophages isindependent of TLR4. This interaction did not depend on TLR2because the phagocytosis of avirulent C. burnetii organisms was

FIGURE 5. Granuloma expression in TLR4�/� mice. TLR4�/� and wtmice were infected by C. burnetii (5 � 105 organisms), and sacrificed atdifferent times. Granulomas were revealed by histopathology. A, Repre-sentative micrographs of tissue pieces from wt and TLR4�/� mice areshown. B, The number of granulomas in the spleen (left) and the liver(right) was determined microscopically. The results are expressed as thenumber of granuloma per square millimeter of tissue, and are the mean �SE of five mice per time point. �, p � 0.05 represents the comparisonbetween wt and TLR4�/� mice. C, The number of cells within the gran-ulomas and the granuloma area were calculated by image analysis of liversections at day 7 postinfection. The results are the mean � SE of five mice.

FIGURE 6. Cytokine production in TLR4�/� mice. Splenocytes andperitoneal macrophages from wt and TLR4�/� mice were isolated fromuninfected (day 0) and C. burnetii-infected mice. Cells were incubated withC. burnetii (10:1 bacterium to cell ratio) for 24 h at 37°C. A, IFN-� wasassayed in splenocyte supernatants by immunoassay. B and C, TNF andIL-10 were assayed in macrophage supernatants by immunoassays. Resultsare expressed as picogram per milliliter and represent the mean � SE ofthree experiments. �, p � 0.05 represents the comparison between wt andTLR4�/� mice.

3701The Journal of Immunology

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

similar in TLR2�/� and wt macrophages. This finding is distinctfrom reports in which atypical LPS from Porphyromonas gingi-valis or Leptospira interrogans are recognized by TLR2 but not byTLR4 (31, 32). Nevertheless, TLR2 may compensate the lack ofTLR4 for C. burnetii phagocytosis. Indeed, PG, known to specif-ically interact with TLR2, increased C. burnetii phagocytosis byTLR4�/� macrophages to the level found in wt macrophages. Li-popeptide, another ligand of TLR2, was recently shown to increasethe phagocytosis of opsonized latex beads in neutrophils (33). Wealso found that C. burnetii-stimulated reorganization of F-actin inmacrophages is mediated by TLR4. Indeed, in TLR4�/� macro-phages, virulent organisms and phase I LPS were unable to stim-ulate the formation of lamellipodia and filopodia. The role ofTLR4 in cytoskeleton reorganization has been previously evokedin macrophages from C3H/HeJ mice stimulated by E. coli LPS in

which the disruption of microfilament network was prevented (34).The mechanisms of TLR4-mediated F-actin reorganization re-mains hypothetical, but it may be related to the activation of src-related kinases because it has been shown that C. burnetii-stimu-lated F-actin reorganization depends on src kinase activation (15).

Although initial events of C. burnetii infection of macrophagesdepend on TLR4, the survival of C. burnetii does not. Virulent C.burnetii survived in macrophages from wt and TLR4�/� mice, andavirulent organisms were cleared by both types of macrophages.This finding suggests that TLR4 is dispensable for C. burnetii sur-vival. We recently showed that the survival of C. burnetii in hu-man monocytes is associated with impaired phagosome maturation(16), but the role of TLRs in intracellular traffic of microorganismsis largely ignored. It has been only suggested that TLR2 is re-cruited to phagosomes containing yeasts (35) and IgG-coated E(36). In murine macrophages from wt and TLR4�/� mice, phago-somes containing virulent C. burnetii did not acquire cathepsin Din contrast to phagosomes containing avirulent organisms. Thisextends to murine macrophages the impairment of phagosome-lysosome fusion reported in human monocytes (16). In addition,phagosomes containing virulent or avirulent organisms acquiredLamp-1, demonstrating the integrity of upstream traffic as de-scribed in human monocytes (16). Hence, the lack of TLR4 had noeffect on phagosome maturation in murine macrophages infectedwith C. burnetii. We also found that the survival of C. burnetii invivo does not involve TLR4. Indeed, the i.p. injection of C. bur-netii into mice leads to the accumulation of bacteria in spleen andliver followed by their clearance in a similar way in wt andTLR4�/� mice. The role of TLR4 in infections caused by intra-cellular pathogens has been poorly documented. Hence, C3H/HeJmice initially control Mycobacterium tuberculosis infection butthey succumb later (37). In mice with TLR4 mutation, the earlyclearance of Ehrlichia chaffeensis is suppressed but the subsequentresistance is preserved (38). Infection with Legionella pneumo-phila proceeds in an identical way in TLR4�/� and wt mice (39).These results suggest that TLR4 is not critical for the clearance ofintracellular microorganisms including C. burnetii.

The immune host response to C. burnetii infection depends onTLR4. Indeed, C. burnetii infection results in the formation ofgranulomas in spleen and liver, which has been associated withprotective immune response (40). In TLR4�/� mice, the expres-sion of granulomas in spleen was more transient than in wt mice,and the number of granulomas in liver was decreased as comparedwith wt mice. In addition, the cell density of granulomas was lowerin infected TLR4�/� mice than in wt mice. The mechanisms ofaltered formation of granulomas are likely multiple. They mayinvolve changes in granuloma cell composition or impaired cyto-kine production necessary to granuloma formation. Hence, in miceinfected with M. tuberculosis, the lack of TLR4 is associated withincreased number of granulomas and increased influx of neutro-phils in granulomas (37). However, the cellular composition ofgranulomas in mice infected with C. burnetii was similar inTLR4�/� and wt mice. The mechanisms leading to granulomaformation involve the production of inflammatory cytokines in-cluding IFN-� and TNF as demonstrated by knockout mice (41–43). We showed in this study that C. burnetii-stimulated produc-tion of IFN-� by splenocytes was lower in TLR4�/� mice than inwt mice. Such results suggest that TLR4 is involved in C. burnetii-induced granuloma formation via IFN-� production. This is dis-tinct from Abel et al. (37) report in which M. tuberculosis-infectedC3H/HeJ mice express more granuloma than C3H/HeN mice with-out modulation of IFN-� production. Similarly, C. burnetii-stim-ulated production of TNF was down-modulated in TLR4�/� miceas compared with wt mice. This finding agrees with some previous

FIGURE 7. TLR2 and macrophage responses to C. burnetii. A and B,Macrophages (2 � 105 cells/assay) from wt and TLR2�/� mice were in-cubated with virulent and avirulent C. burnetii (200:1 bacterium to cellratio) for 4 h (A) and 10 min (B). A, Bacteria were detected by immuno-fluorescence. The results are expressed as phagocytosis index and are themean � SE of three experiments. B, F-actin was labeled with 10 U/mlbodipy phallacidin, and cells were examined by laser scanning confocalmicroscopy. The results are expressed as the percentage of macrophagesshowing filopodia and represent the mean � SE of three experiments. C,Macrophages (2 � 105 cells/assay) from wt and TLR4�/� mice were pre-treated by PG (50 ng/ml) for 30 min and incubated with virulent C. burnetii(200:1 bacterium to cell ratio) for 4 h at 37°C. Bacteria were detected byimmunofluorescence. The results are expressed as phagocytosis index andare the mean � SE of three experiments. �, p � 0.05 represents the com-parison of C. burnetii uptake by TLR4�/� macrophages in the presenceand the absence of PG.

3702 ROLE OF TLR4 IN C. burnetii PHAGOCYTOSIS AND HOST RESPONSE

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

reports in which TNF production induced by soluble LPSs, Gram-negative bacteria and M. tuberculosis was dramatically impairedwhen TLR4 is lacking (37). The results concerning impaired pro-duction of IFN-� and TNF in TLR4�/� mice are consistent withthe hypothesis that the engagement of TLR4 leads to the produc-tion of type 1 cytokines required for protection against intracellularmicroorganisms, in contrast to TLR2 engagement that favors theproduction of type 2 cytokines (2, 44). The down-modulation ofgranuloma formation does not result from increased production ofcytokines known to impair the production of type 1 cytokines suchas IL-10. Indeed, the production of IL-10 was markedly impairedin TLR4�/� mice. This finding suggests that TLR4 is necessaryfor C. burnetii-stimulated cytokine production.

We reported in this study that LPS from an obligate intracellularorganism such as C. burnetii required TLR4 to transduce signals inhost cells. Indeed, TLR4 was involved in the uptake of virulent C.burnetii by macrophages and C. burnetii-stimulated F-actin reor-ganization. However, TLR4 did not control the microbicidal ac-tivity of macrophages toward C. burnetii and, in vivo, tissue in-fection and bacterial clearance. TLR4 was in contrast necessary forthe formation of protective granulomas and the production ofIFN-� and TNF. Hence, TLR4 likely plays a critical role in theearly responses of the host to C. burnetii infection by enabling itto develop protective immune responses, but the control of latephases of the infection requires other mechanisms.

AcknowledgmentsWe acknowledge G. Grau and S. Dumler for their critical reading of themanuscript.

References1. Medzhitov, R., and C. A. Janeway. 2002. Decoding the patterns of self and

nonself by the innate immune system. Science 296:298.2. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol.

1:135.3. Vasselon, T., and P. A. Detmers. 2002. Toll receptors: a central element in innate

immune responses. Infect. Immun. 70:1033.4. Mege, J. L., M. Maurin, C. Capo, and D. Raoult. 1997. Coxiella burnetii: the

’query’ fever bacterium: a model of immune subversion by a strictly intracellularmicroorganism. FEMS Microbiol. Rev. 19:209.

5. Hackstadt, T. 1986. Antigenic variation in the phase I lipopolysaccharide of Cox-iella burnetii isolates. Infect. Immun. 52:337.

6. Amano, K., J. C. Williams, S. R. Missler, and V. N. Reinhold. 1987. Structureand biological relationships of Coxiella burnetii lipopolysaccharides. J. Biol.Chem. 262:4740.

7. Vodkin, M. H., J. C. Williams, and E. H. Stephenson. 1986. Genetic heteroge-neity among isolates of Coxiella burnetii. J. Gen. Microbiol. 132:455.

8. Hoover, T. A., D. W. Culp, M. H. Vodkin, J. C. Williams, and H. A. Thompson.2002. Chromosomal DNA deletions explain phenotypic characteristics of twoantigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii NineMile strain. Infect. Immun. 70:6726.

9. Gajdosova, E., E. Kovacova, R. Toman, L. Skultety, M. Lukacova, and J. Kazar.1994. Immunogenicity of Coxiella burnetii whole cells and their outer membranecomponents. Acta Virol. 38:339.

10. Williams, J. C., and D. M. Waag. 1991. Antigens, virulence factors, and biolog-ical response modifiers of Coxiella burnetii: strategies for vaccine development.In The Biology of Coxiella burnetii. J. C. Williams and H. A. Thompson, eds.CRC Press, Boca Raton, p. 175.

11. Tujulin, E., B. Lilliehook, A. Macellaro, A. Sjostedt, and L. Norlander. 1999.Early cytokine induction in mouse P388D1 macrophages infected by Coxiellaburnetii. Vet. Immunol. Immunopathol. 68:159.

12. Dellacasagrande, J., E. Ghigo, S. Machergui-Hammami, R. Toman, D. Raoult,C. Capo, and J. L. Mege. 2000. �v�3 integrin and bacterial lipopolysaccharide areinvolved in Coxiella burnetii-stimulated production of tumor necrosis factor byhuman monocytes. Infect. Immun. 68:5673.

13. Capo, C., F. P. Lindberg, S. Meconi, Y. Zaffran, G. Tardei, E. J. Brown,D. Raoult, and J. L. Mege. 1999. Subversion of monocyte functions by Coxiellaburnetii: impairment of the cross-talk between �v�3 integrin and CR3. J. Immu-nol. 163:6078.

14. Meconi, S., V. Jacomo, P. Boquet, D. Raoult, J. L. Mege, and C. Capo. 1998.Coxiella burnetii induces reorganization of the actin cytoskeleton in humanmonocytes. Infect. Immun. 66:5527.

15. Meconi, S., C. Capo, M. Remacle-Bonnet, G. Pommier, D. Raoult, andJ. L. Mege. 2001. Activation of protein tyrosine kinases by Coxiella burnetii: rolein actin cytoskeleton reorganization and bacterial phagocytosis. Infect. Immun.69:2520.

16. Ghigo, E., C. Capo, C. H. Tung, D. Raoult, J. P. Gorvel, and J. L. Mege. 2002.Coxiella burnetii survival in THP-1 monocytes involves the impairment ofphagosome maturation: IFN-� mediates its restoration and bacterial killing. J. Im-munol. 169:4488.

17. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda,and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:443.

18. Ghigo, E., C. Capo, M. Aurouze, C. H. Tung, J. P. Gorvel, D. Raoult, andJ. L. Mege. 2002. Survival of Tropheryma whipplei, the agent of Whipple’sdisease, requires phagosome acidification. Infect. Immun. 70:1501.

19. Toman, R., and L. Skultety. 1996. Structural study on a lipopolysaccharide fromCoxiella burnetii strain Nine Mile in avirulent phase II. Carbohydr. Res. 283:175.

20. Toman, R., L. Skultety, P. Ftacek, and M. Hricovini. 1998. NMR study ofvirenose and dihydrohydroxystreptose isolated from Coxiella burnetii phase Ilipopolysaccharide. Carbohydr. Res. 306:291.

21. Pizarro-Cerda, J., E. Moreno, V. Sanguedolce, J. L. Mege, and J. P. Gorvel. 1998.Virulent Brucella abortus prevents lysosome fusion and is distributed withinautophagosome-like compartments. Infect. Immun. 66:2387.

22. La Scola, B., H. Lepidi, and D. Raoult. 1997. Pathologic changes during acute Qfever: influence of the route of infection and inoculum size in infected guineapigs. Infect. Immun. 65:2443.

23. Lepidi, H., P. E. Fournier, and D. Raoult. 2000. Quantitative analysis of valvularlesions during Bartonella endocarditis. Am. J. Clin. Pathol. 114:880.

24. Tissot Dupont, H., X. Thirion, and D. Raoult. 1994. Q fever serology: cutoffdetermination for microimmunofluorescence. Clin. Diagn. Lab. Immunol. 1:189.

25. Poltorak, A., P. Ricciardi-Castagnoli, S. Citterio, and B. Beutler. 2000. Physicalcontact between lipopolysaccharide and Toll-like receptor 4 revealed by geneticcomplementation. Proc. Natl. Acad. Sci. USA 97:2163.

26. Wright, S. D., R. A. Ramos, A. Hermanowski-Vosatka, P. Rockwell, andP. A. Detmers. 1991. Activation of the adhesive capacity of CR3 on neutrophilsby endotoxin: dependence on lipopolysaccharide-binding protein and CD14.J. Exp. Med. 173:1281.

27. Chang, S., K. J. Stacey, J. Chen, E. O. Costelloe, A. Aderem, and D. A. Hume.1999. Mechanisms of regulation of the MacMARCKS gene in macrophages bybacterial lipopolysaccharide. J. Leukocyte Biol. 66:528.

28. Hynes, R. O. 2002. Integrins: bidirectional, allosteric signaling machines. Cell110:673.

29. Williams, L. M., and A. J. Ridley. 2000. Lipopolysaccharide induces actin reor-ganization and tyrosine phosphorylation of Pyk2 and paxillin in monocytes andmacrophages. J. Immunol. 164:2028.

30. Capo, C., A. Moynault, Y. Collette, D. Olive, E. J. Brown, D. Raoult, andJ. L. Mege. 2003. Coxiella burnetii avoids macrophage phagocytosis by inter-fering with spatial distribution of complement receptor 3. J. Immunol. 170:4217.

31. Hirschfeld, M., J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody,D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel. 2001. Signaling byToll-like receptor 2 and 4 agonists results in differential gene expression in mu-rine macrophages. Infect. Immun. 69:1477.

32. Werts, C., R. I. Tapping, J. C. Mathison, T. H. Chuang, V. Kravchenko,I. Saint Girons, D. A. Haake, P. J. Godowski, F. Hayashi, A. Ozinsky, et al. 2001.Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mech-anism. Nat. Immun. 2:346.

33. Hayashi, F., T. K. Means, and A. D. Luster. 2003. Toll-like receptors stimulatehuman neutrophil function. Blood 102:2660.

34. Wonderling, R. S., A. Ghaffar, and E. P. Mayer. 1996. Lipopolysaccharide-in-duced suppression of phagocytosis: effects on the phagocytic machinery. Immu-nopharmacol. Immunotoxicol. 18:267.

35. Underhill, D. M., A. Ozinsky, A. M. Hajjar, A. Stevens, C. B. Wilson,M. Bassetti, and A. Aderem. 1999. The Toll-like receptor 2 is recruited to mac-rophage phagosomes and discriminates between pathogens. Nature 401:811.

36. Ozinsky, A., D. M. Underhill, J. D. Fontenot, A. M. Hajjar, K. D. Smith,C. B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for patternrecognition of pathogens by the innate immune system is defined by cooperationbetween Toll-like receptors. Proc. Natl. Acad. Sci. USA 97:13766.

37. Abel, B., N. Thieblemont, V. J. Quesniaux, N. Brown, J. Mpagi, K. Miyake,F. Bihl, and B. Ryffel. 2002. Toll-like receptor 4 expression is required to controlchronic Mycobacterium tuberculosis infection in mice. J. Immunol. 169:3155.

38. Ganta, R. R., M. J. Wilkerson, C. Cheng, A. M. Rokey, and S. K. Chapes. 2002.Persistent Ehrlichia chaffeensis infection occurs in the absence of functional ma-jor histocompatibility complex class II genes. Infect. Immun. 70:380.

39. Lettinga, K. D., S. Florquin, P. Speelman, R. van Ketel, T. van der Poll, andA. Verbon. 2002. Toll-like receptor 4 is not involved in host defense againstpulmonary Legionella pneumophila infection in a mouse model. J. Infect. Dis.186:570.

40. Maurin, M., and D. Raoult. 1999. Q fever. Clin. Microbiol. Rev. 12:518.41. Kindler, V., A. P. Sappino, G. E. Grau, P. F. Piguet, and P. Vassalli. 1989. The

inducing role of tumor necrosis factor in the development of bactericidal gran-ulomas during BCG infection. Cell 56:731.

42. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses tointerferon-�. Annu. Rev. Immunol. 15:749.

43. Bean, A. G., D. R. Roach, H. Briscoe, M. P. France, H. Korner, J. D. Sedgwick,and W. J. Britton. 1999. Structural deficiencies in granuloma formation in TNFgene-targeted mice underlie the heightened susceptibility to aerosol Mycobacte-rium tuberculosis infection, which is not compensated for by lymphotoxin. J. Im-munol. 162:3504.

44. Re, F., and J. L. Strominger. 2001. Toll-like receptor 2 (TLR2) and TLR4 dif-ferentially activate human dendritic cells. J. Biol. Chem. 276:37692.

3703The Journal of Immunology

by guest on April 11, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from